The present invention relates to method for producing polyhydroxyalkanoates in recombinant organisms.
Plastic materials have become an integral part of contemporary life because they possess many desirable properties, including durability and resistance to degradation. Over the past 10-20 years, their widespread use have been increasingly regarded as a source of environmental and waste management problems. Industrial societies are now more aware of the impact of discarded plastic on the environment, and of their deleterious effect on wildlife and the aesthetic qualities of cities and forests. Problems associated with the disposal of waste and reduction in the availability of landfills have also focused attention on plastics, which accumulate in the environment at a rate of 25 million tonnes per year (Lee, 1996). These problems have created much interest in the development and production of biodegradable plastics. Biodegradable polymers are composed of material which can be degraded either by non-enzymatic hydrolysis or by the action of enzymes secreted by microorganisms. Estimates of the current global market for these biodegradable plastics range up to 1.3 billion kg per year (Lindsay, 1992).
Among the various biodegradable plastics available, there is a growing interest in the group of polyhydroxyalkanoates (PHAs). These are natural polymers produced by a variety of bacteria and they are 100% biodegradable. By changing the carbon source and bacterial strains used in the fermentation processes, PHA-biopolymers having a wide variety of mechanical properties have been produced. Their physical characteristics range from hard crystalline to elastic, depending on the composition of monomer units (Anderson and Dawes, 1990). The majority of PHAs are composed of R-(xe2x88x92)-3-hydroxyalkanoic acid monomers ranging from 3 to 14 carbons in length (C3-C14). The simplest member of the family, P(3HB) (C4), is highly crystalline, relatively stiff, and becomes brittle over a period of days upon storage under ambient conditions (Barham et al., 1984; De Koning et al., 1992; Doi, 1990; Holmes, 1988). Therefore, attempts have been made to decrease the brittleness of P(3HB) either by incorporating comonomers such as P(3HV), by blending with other polymers or blending with chemically synthesized atactic P(3HB) (Holmes, 1988; Kumagai and Doi, 1992 a, 1992 b, 1992 c; Pearce and Marchessault, 1994).
The P(3HB-co-3HV) copolymer, developed by ZENECA under the tradename BIOPOL(trademark), has improved mechanical properties compared to P(3HB). As the fraction of P(3HV) (C5) increases, the polymer becomes tougher, more flexible and have an higher elongation to break (Doi et al., 1990). The medium-chain-length (MCL) PHAs are semicrystalline elastomers with a low melting point, low tensile strength, and high elongation to break. They thus have physico-chemical characteristics that make them more appealing than homogeneous P(3HB); they can even be used as a biodegradable rubber after cross linking by electron-beam irradiation (De Koning et al., 1994; Gagnon et al., 1992; Gross et al., 1989; Preusting et al., 1990).
PHAs have been shown to occur in over 90 genera of Gram-positive and Gram-negative bacteria species (Steinbxc3xcchel, 1991). Over 40 different PHAs have been characterized, with some polymers containing unsaturated bonds or various functional groups (Steinbxc3xcchel, 1991). Bacteria synthetise and accumulate PHAs as carbon and energy storage materials or as a sink for redundant reducing power under the condition of limiting nutrients in the presence of excess carbon sources (Byrom, 1994; Doi, 1990; Steinbxc3xcchel and Valentin, 1995). When the supply of the limiting nutrient is restored, the PHAs are degraded by intracellular depolymerases and subsequently metabolized as carbon and energy source (Byrom, 1990; Doi, 1990). The monomer 3HAs released from degradation of these microbial polyesters are all in the R-(xe2x88x92)-configuration due to the stereo specificity of biosynthetic enzymes (Anderson and Dawes, 1990). The molecular weights of polymers are in the range of 2xc3x97105 to 3xc3x97106 Daltons, depending on the microorganism and growth condition (Byrom, 1994). PHAs accumulate in the cells as discrete granules, the number per cell and size of which can vary among the different species; 8 to 13 granules per cell of 0.2 to 0.5 xcexcm diameter have been observed in Alcaligenes eutrophus (Byrom, 1994).
PHAs can be subdivided in two groups depending on the number of carbon atoms in the monomer units: short-chain-length-(SCL) PHAs, which contain 3-5 carbon atoms, and medium-chain-length-(MCL) PHAs, which contain 6-14 carbon atoms (Anderson and Dawes, 1990). This is mainly due to the substrate specificity of the PHA synthases that can only accept 3HA monomers of a certain range of carbon lengths (Anderson and Dawes, 1990). The PHA synthase of Alcaligenes eutrophus can polymerize C3-C5 monomers, but not C6 or higher. On the other hand, the PHA synthase of Pseudomonas oleovorans only accepts C6-C14 monomers. Of particular interest is the capacity of some PHA synthase to polymerize 3-hydroxy-, 4-hydroxy- and 5-hydroxy-alkanoates (Steinbxc3xcchel and Schlegel, 1991). Even though most of the PHA synthases examined to date are specific for the synthesis of either SCL- or MCL-PHAs, at least six cases were recently reported in which the bacteria were able to synthesize copolymer consisting of SCL and MCL units (Lee, 1996).
P(3HB) is the most widespread and thoroughly characterized PHA, and most of the knowledge has been obtained from Alcaligenes eutrophus (Steinbxc3xcchel, 1991). In this bacterium, P(3HB) is synthesized from acetyl-CoA by the sequential action of three enzymes (FIG. 1).The first one, 3-ketothiolase, catalyses the reversible condensation of two acetyl-CoA moieties to form acetoacetyl-CoA. Acetoacetyl-CoA reductase subsequently reduces acetoacetyl-CoA to R-(xe2x88x92)-3-hydroxybutyryl-CoA, which is then polymerized by the action of PHA synthase to form P(3HB). A number of PHAs with different C3 to C5 monomers have been produced in A. eutrophus, the nature and proportion of these monomers being influenced by the type and relative quantity of the carbon sources supplied to the growth media (Steinbxc3xcchel and Valentin, 1995). Pseudomonas oleovorans and most pseudomonades belonging to the ribosomal rRNA homology group I synthesize MCL-PHAs from various MCL-alkanes, alkanols, or alkanoates (Steinbxc3xcchel and Valentin, 1995). The composition of PHA produced is related to the substrate used for growth, with the polymer being mostly composed of monomers which are 2n carbons shorter than the substrates used. It was suggested that the acyl-CoA derived from alkanoic acids enter the xcex2-oxidation pathway and R-(xe2x88x92)-3hydroxyacyl-CoA intermediates used by the PHA synthase are generated either through reduction of 3-ketoacyl-CoA by a ketoacyl-CoA reductase, conversion of S-(+)-3hydroxyacyl-CoA normally produced by the pathway to the R-(xe2x88x92)-isomer by an epimerase, or the direct hydration of enoyl-CoA by an enoyl-CoA hydratase (Poirier et al., 1995).
Most pseudomonades belonging to rRNA homology group I, except P. oleovorans, also synthesize MCL-PHAs when grown on substrates non related to fatty acids and alkanoates, such as gluconate, lactate, glycerol, and hexoses (Anderson and Dawes, 1990; Huijberts et al., 1994; Timm and Steinbxc3xcchel, 1990). These substrates must be first converted into acetyl-CoA to be used for the PHAs biosynthesis. This suggests that, in theory, microorganisms, plants and even animals, must be able to synthesize PHA following the transfection of a limited number of genes. In these bacteria, three main pathways have been proposed for the synthesis of PHA precursors (Huijberts et al., 1992, 1994).
(i) A detailed analysis of the composition of PHA produced by P. putida grown on glucose have shown that the monomers are structurally identical to the acyl-moieties of the 3-hydroxyacyl-ACP intermediates of the novo fatty acid biosynthesis. Since it has not been shown that PHA synthase can accept acyl-ACPs as substrates, these must therefore be converted to acyl-CoAs by a transacylase before entering the PHA pathway.
(ii) Fatty acid degradation by xcex2-oxidation is the main pathway when fatty acids are used as substrate.
(iii) It has been found that some of the monomeric units of PHA are one C2 unit longer than the fatty acid used as substrate. Chain elongation by condensation of an acetyl-CoA to the acyl-CoA has therefore been suggested.
A complex picture thus emerges in which the steps linking the different pathways implied and PHA synthesis are at present unknown (FIG. 2). It is assumed, but not demonstrated, that the ultimate substrate for polymerization is the R form of the CoA-activated 3-hydroxy fatty acid intermediates. Expression of the synthases of P. putida in wild-type E. coli is not sufficient to produce PHA in this bacterium (Huisman, 1991). More genetic information from Pseudomonas spp. seems to be needed to enable PHA synthesis in Escherichia coli. We can speculate that the missing step in prokaryotic organisms other than pseudomonades is the formation of R-(xe2x88x92)-3-OH-acyl-CoA of more than 5C.
The bio(techno)logical approach for the production of PHAs use microbial systems. The major commercial drawback of the so-produced bacterial PHAs are their high production cost, making them substantially more expensive than synthetic plastics. At present, Zeneca produces approximately 1,000 tons per year of P(3HB-co-3HV) copolymer at a cost of approximately $16/kg. At a production rate of 10,000 tons per year or more, the most optimistic scenario would put the cost at $5/kg. With the cost of many synthetic plastics such as polypropylene and polyethylene, being less than $1/kg, PHA appear too costly for most low-value consumer products (Poirier et al., 1995).
Engineering of novel pathways in eucaryotic cell systems seems to be a beneficial alternative to the production of PHAs in bacteria. On one hand, yeast and insect cells can be used as models to gain information on PHAs synthesis in eucaryotes (Hahn et al., 1996; Sherman, 1996). On the other hand, a new possibility for the production of PHAs on a large scale and at costs comparable to synthetic plastics has arisen from the demonstration of their production in transgenic plants (Poirier et al., 1992). Production of PHA on an agronomic scale could allow synthesis of biodegradable plastics in the million ton scale compared to fermentation which produces material in the thousand ton scale (Poirier et al., 1995). In addition, plant production of PHAs would use carbon dioxide, water and sunlight as raw materials to produce PHA in an environmentally friendly and sustainable manner.
Synthesis of PHB in plants was initially explored by expression of the PHB biosynthetic genes of A. eutrophus in the plant Arabidopsis thaliana (Poirier et al., 1992). Although of no agricultural importance, this small oil seed plant was chosen for its extensive use as a model system for genetic and molecular studies in plants. These plants accumulated P(3HB) granules that were 0.2 to 0.5 xcexcm in diameter in the nucleus, vacuole, and cytoplasm. However, the amount of P(3HB) accumulated was only 100 xcexcg/g fresh weight. Furthermore, plants were impaired in their growth, probably due to the severe deviation of substrate from the mevalonate pathway which is essential for chlorophyll synthesis.
To avoid this problem and to improve polymer accumulation, further genetic manipulation have been carried out to divert reduced carbon away from endogenous metabolic pathways and to regulate the tissue specificity and timing of gene expression. The plastid was suggested to be the ideal location for P(3HB) accumulation because it is the location of high flux of carbon through acetyl-CoA. Genetically engineered genes of A. eutrophus were then successfully targeted to the plant plastids, where the enzymes were active (Nawrath et al., 1995). The A. eutrophus PHA biosynthesis genes were modified for plastid targeting by fusing the transit peptide of ribulose biphosphate carboxylase to their N-terminal ends and were put under the control of the constitutive CaMV 35S promoter. The hybrid expressing the A. eutrophus PHA synthesis enzymes accumulated P(3HB) up to 10 mg/g fresh weight, representing ca. 14% of dry weight.
The knowledge acquired in this study is not only useful to optimise strategies for the production of PHB in recombinant organisms, but could also be used for the production of PHAs other than PHB, for example MCL-PHAs. For plant production of PHAs to become commercially viable, the genes must be transfected into a suitable plant species which has the agronomic properties to provide high yields of PHA per hectare, at unlimited scale and at economic prices. Subcellular localization signals and promoters must be chosen which allow the enzymes utilized to intercept the desired plant metabolites for incorporation into the polymer.
Different strategies have been proposed for production of PHAs in plants. Substitution of cytoplasmic oil bodies by PHA granules, production of PHAs in glyoxysomes or production of PHAs in leucoplasts have been proposed to be carried out in lipid-accumulating tissues of oilseed crops, such as seed endosperm or fruit mesocarp (van der Leij and Witholt, 1995; Hahn et al., 1996; Srienc and Leaf, 1996). In this tissue, triglycerides provide energy and carbon for germination of the new plant before establishment of photosynthesis. In contrast, PHAs would not be degraded in plants because of the absence of endogenous enzymes capable of hydrolyzing the polymer. Interfering with synthesis and degradation of fatty acids, in respectively plastids and glyoxysomes, by diverting energy into PHAs in this stage of development is likely to impair germination and/or seedling growth. As a post-harvest event this can be desirable. However, this inherent characteristic of the proposed strategy will create problems in the production of viable hybrid seeds. The expression of the enzymes during germination should be restricted to the second generation of seeds or fruit. For this, solutions will have to be found. It is probable that controlled expression of these genes will necessitate the use of promoters stimulated by external signals (Williams et al., 1992).
Plastids are regarded as the most amenable targets for PHA production. The production in chloroplasts, directly coupled to the novo fatty acid synthesis has many advantages. First, every important crop can be used. Second, in leaves, fatty acid metabolism is not as important as in seeds and targeting to this tissue is not likely to impair growth of the plant. Third, it is the most direct way for PHAs production, since the plant does not have to produce long-chain fatty acids or triglycerol before diverting fatty acid degradation products into PHAs, like in the case of glyoxysomal degradative mechanisms. Fourth, as shown for the synthesis of PHB, compartmentalization in plastids does not impair growth and then appear to be favoured over unrestricted synthesis in the cytosol.
Pseudomonas aeruginosa belongs to the group of pseudomonades of the rRNA homology group I that synthesize MCL-PHAs when grown either on alkanes or on unrelated substrates such as gluconate (Timm and Steinbxc3xcchel, 1990). A PHA synthase locus in P. aeruginosa was identified by the use of a 32P-labeled 30-mer synthetic oligonucleotide probe, whose sequence design was based on that of a highly conserved region of PHA synthases in A. eutrophus and P. oleovorans (Steinbxc3xcchel et al., 1992). The organization of the locus consist of two genes coding for PHA synthases (phaC1, phaC2) separated by a gene coding for a putative PHA depolymerase (phaD), and a fourth gene (ORF3) downstream of phaC2 with an unknown function (Timm and Steinbxc3xcchel, 1992). It has been shown that these synthases are similar to those found in P. oleovorans, who is unable to synthesize MCL-PHAs from unrelated substrates (Huijberts et al., 1992).
As was shown in P. aeruginosa, intermediates of fatty acid biosynthesis and xcex2-oxidation are likely to contribute to the formation of PHA polymers. It is most likely that the intermediate precursors to PHA synthesis are either ketoacyl-CoA, S-(+)-3-OH-acyl-CoA, enoyl-CoA, or R-(xe2x88x92)-3-OH-acyl-ACP. However, since substrate specificity for PHA synthase has not yet been thoroughly tested, it is still unclear whether this enzyme could accept other derivative forms of 3-hydroxyacyl moieties, like for instance ACP derivatives. This can be of substantial impact on the choice of the best strategy for production in recombinant organisms: if the recombinant enzyme can accept, even at sub-optimum rates, ACP derivatives as substrate, then its targeting to chloroplasts would be the only required engineering alteration needed to induce PHA accumulation in leaf cells.
Monomeric units of PHAs, as it is the case for these of PHBs, are of the isomeric form R-(xe2x88x92)-; this has been repeatedly demonstrated by the analysis of hydrolysate from PHA granules. Enzymologic analysis also show that PHB synthases have a definite specificity for R-(xe2x88x92)-3-OH-acyl-CoA as substrates. Although the substrate specificity of PHA synthases has not yet been thoroughly characterized with purified enzyme preparations, their high homology with PHB syntheses and the analysis of their reaction product strongly suggest that they share a preference for R-(xe2x88x92)-3-OH-acyl CoA substrates with PHB synthases.
There are no demonstration of a metabolic pathway that would supply monomeric subunits to the polymerization reaction in Pseudomonades, nor in any other organisms. Known degradation pathways starting with acyl-CoAs produce S-(xe2x88x92)3-OH-acyl-CoAs and synthetic pathways produce R-(xe2x88x92)-acyl-ACPs, none of which can serve as substrate for the PHA synthesis reaction.
As further background, the following U.S. Patent should be reviewed: 5,650,555; 5,502,273; 5,245,023; 5,610,041; 5,229,279; 5,534,432; 5,750,848; 5,663,063; 5,480,794; 5,750,848; 5,801,027; 5,298,421 and 5,250,430.
This invention is directed at the production of polyhydroxyalkanoates in recombinant organisms, through the engineering of a new metabolic pathway which produces R-(xe2x88x92)-3-OH-acyl-CoAs monomeric subunits of adequate length to serve as substrates for the activity of PHA synthases.
More specifically, it describes the methodology that is used to produce transgenic organism with a new metabolic pathway that partially deviates fatty acids from their normal synthetic pathways, towards the formation of R-(xe2x88x92)-3-OH-acyl-CoAs that serves as substrates for the synthesis of hydroxyalkanoate polymers in chloroplasts.
The engineered synthetic metabolic pathway of the present invention initially produces free (C8) fatty acids from the fatty acid synthesis pathway through the action of a thioesterase, that will then add a CoA moiety to the free fatty acid through the action of an acyl-CoA synthase, that will produce 3-(xe2x88x92)-ketoacyl-CoAs from the acyl-CoA through the action of athiolase, that will produce R-(xe2x88x92)-OH-acyl-CoAs from the 3-keto acid-CoAs through the action of a unique dehydrogenase isoform from yeast. These R-(xe2x88x92)-3-OH-acyl-CoAs will finally be used as substrate for the PHA synthase reaction.
Thus according to the present invention there is provided a method for the production of polyhydroxyalkanoates comprising: selecting a transgenic organism comprising a foreign DNA sequence encoding an enzyme having dehydrogenase activity, which will produce a R-(xe2x88x92)-hydroxyacyl-CoA from a keto acid-CoA, wherein said R-(xe2x88x92)-hydroxyacyl-CoA will serve as a substrate for polyhydroxyalkanoate synthase; and producing said polyhydroxyalkanoate.
Further, according to the present invention there is provided a method for producing a polyhydroxyalkanoate in a host comprising: selecting a host for expression of genes encoding enzymes required for synthesis of a polyhydroxyalkanoate; introducing into said host structural genes encoding enzymes selected from the group consisting of: a thioesterase, an acyl-CoA synthetase, a thiolase, a hydroxyacyl-CoA dehydrogenase, and a polyhydroxyalkanoate synthase; expressing the enzymes encoded by the genes; and providing the appropriate substrates for the expressed enzymes to synthesis the polyhydroxyalkanoate.
According to the present invention there is also provided a cloning vector comprising foreign DNA encoding an enzyme having dehydrogenase activity, which will produce R-(xe2x88x92)-hydroxyacyl-CoA from a keto acid-CoA. According to one embodiment of the invention the cloning vector further comprises a DNA sequence encoding an enzyme having thioesterase activity; an enzyme having acyl-CoA synthetase activity; an enzyme having thiolase activity; and an enzyme having polyhydroxyalkanoate synthase activity.
According to the present invention there is also provided a host cell comprising foreign DNA encoding an enzyme having dehydrogenase activity, which will produce R-(xe2x88x92)-hydroxyacyl-CoA from a keto acid-CoA. According to one embodiment of the invention the host cell further comprises a DNA sequence encoding an enzyme having thioesterase activity; an enzyme having acyl-CoA synthetase activity; an enzyme having thiolase activity; and an enzyme having polyhydroxyalkanoate synthase activity.
According to the present invention there is also provided a transgenic organism comprising foreign DNA encoding an enzyme having dehydrogenase activity, which will produce R-(xe2x88x92)-hydroxyacyl-CoA from a keto acid-CoA. According to one embodiment of the invention the transgenic organism further comprises a DNA sequence encoding an enzyme having thioesterase activity; an enzyme having acyl-CoA synthetase activity; an enzyme having thiolase activity; and an enzyme having polyhydroxyalkanoate synthase activity. In one example of this embodiment the transgenic organism is a plant.